Anti-leishmanial activity of betel leaf extract

ABSTRACT

This invention relates to method of treating visceral leishmaniasis or kala-azar by administering effective amount of betel leaf extract or lyophilized extract together with or associated with an additive and a composition comprising betel leaf extract with a pharmaceutically acceptable additive.

FIELD OF INVENTION

[0001] This invention relates to the treatment of leishmaniasis inanimals including human beings, preferably, the extract of betel leaf isused for the treatment of leishmaniasis in human beings. Chemotherapyfor visceral leishmaniasis has seen little progress in recent years.Antileishmanicidal activity of betel leaf extracts suggests itspotential use to treat human visceral leishmaniasis.

BACKGROUND AND PRIOR ART REFERENCES

[0002] Leishmaniasis commonly known as kala-azar as known in India is aglobal health problem. Infection by various species and strains ofLeishmania causes a wide spectrum of disease in humans, with manydifferent clinical presentations. The severity of the disease is largelydictated by the immunological status of the infected individual and bythe species of Leishmania involved. Approximately 350 million people in80 countries are estimated to be threatened by the disease. The WorldHealth Organization (WHO) estimated 12 million cases of leishmaniasisworldwide, with over 400,000 new cases each year. The visceral form ofleishmaniasis, or kala-azar, is caused by the parasite Leishmaniadonovani and is often fatal. Despite tremendous progress made inunderstanding the biochemistry and molecular biology of Leishmaniaspecies, treatment by chemotherapy has seen very little progress inrecent years. The toxic pentavalent antimonials remain the mainstay oftreatment for leishmaniasis. The second line drugs, pentamidine andamphotericin B, although used clinically, have serious toxic sideeffects. Therefore, improved drug therapy for leishmaniasis remainsdesirable. Indolylquinoline derivatives have recently been showncytotoxic to Leishmania donovani promastigotes and amastigotes in vitroand are effective in treating murine visceral leishmaniasis (GanesChakrabarti, Anirban Basu, Partha Pratim Manna, Sashi Bhusahan Mahato,Nirup Bikash Mandal and Santu Bandyopadhyay, Journal of AntimicrobialChemotherapy (1999) Vol. 43 pp.359-366).

[0003] Betel leaves have a strong pungent aromatic flavor and are widelyused as a masticatory. Generally, mature or over mature leaves, whichhave ceased growing but not yet become brittle are used for chewing. Thebasic preparation for chewing purposes consists of betel leaf smearedwith hydrated lime and catechu to which scrapings of arecanut are added;flavorings such as coconut shavings, clove, cardamom, fennel, powderedliquorice, nutmeg and also tobacco are used according to one's taste. Insome places prepared pan is covered with silver or gold leaf. As amasticatory, it is credited with many properties: it is aromatic,digestive, stimulant and carminative. Medicinally, it is useful incatarrhal and pulmonary affections; it is also used for poultices. Theeffects of chewing of betel with arecanut and other adjuncts are theexcitation of the salivary glands and the irritation of the mucousmembrane of the mouth. The red coloration produced is due to a pigmentin the arecanut, which manifests itself under the action of alkali inlime and catechu. A mild degree of stimulation is produced, resulting ina sensation of warmth and well being, besides imparting a pleasant odor.The most important factor determining the aromatic value of the leaf isthe amount and particularly the nature of the essential oil present.Betel leaves from different regions vary in smell and taste. The mostpungent is the Sanchi type, while the most mild and sweet ones are fromMadras. The betel leaves contain essential oils, the content of oilvaries from 0.7 to 2.6 per cent depending upon the varieties of leaves.The oil consists of phenols and terpens. The higher the proportion ofphenol oil, the better the quality. An isomer of eugenol namedchavibetol (betel phenol; 4-allyl-2-hydroxy-1-methoxy benzene) isconsidered to be the characteristic constituent of betel oil. It ishowever, absent in Indian samples. Betel oil of Indian types contain asa predominant phenolic constituent. Oil of betel has been used in thetreatment of various respiratory catarrhs, as a local application eitherby gargle or by inhalation in diphtheria. It has carminative properties.It exhibits in different action on the central nervous system ofmammals; lethal doses produce deep narcosis leading to death within afew hours. The essential oil and extracts of the leaves possess activityagainst several Gram-positive and Gram-negative bacteria such asMicrococcus pyogenes var. albus and var. aureus, Bacillus subtilis andB. megaterium, Diplococcus pneumoniae, Streptococcus pyogenes,Escherichia coli, Salmonella typhosa, Vibrio comma, Shigelladysenteriae, Proteus vulgaris, Pseudomonas solanacaerum, Sarcina luteaand Erwinia carotovora. The essential oil and leaf extracts also showedantifungal activity against Asperigillus niger and A. oryzae, Curvularialunata and Fusarium oxysporum. The oil is found to be lethal in about 5minutes to the protozoa Paramaecium caudatum (Wealth of India, Vol.8,pg.84-94).

OBJECTS OF THE INVENTION

[0004] The main object of the invention is to provide a method oftreating animals including human beings for leishmaniasis by the way ofadministering betel leaf extract or lyophilized betel leaf extract.

[0005] Another object of the present invention is to provide acomposition comprising betel leaf extract, which is useful for thetreatment of leishmaniasis of human being.

[0006] Yet another object of the present invention is the preparation ofthe betel leaf water extract.

SUMMARY OF THE INVENTION

[0007] To meet the above objects, the invention provides use of betelleaf extract for the treatment of leishmaniasis in human beings andanimals.

DETAILED DESCRIPTION

[0008] The present invention relates to a composition comprising betelleaf extract, which is useful for the treatment of leishmaniasis ofhuman being and a process for the preparation of the betel leaf waterextract. This invention also provides a method of treating the humanbeing for leishmaniasis by the way of administering the extract orlyophilized extract as a composition comprising betel leaf extract alongwith a pharmaceutically acceptable additive.

[0009] Accordingly the invention provides a pharmaceutical compositionuseful for the treatment of visceral leishmaniasis, or kala-azar asknown in India, said composition comprising effective amount of betelleaf extract or lyophilized extract together with or associated with apharmaceutically acceptable additive.

[0010] In another embodiment the additive is selected in such a mannerthat does not interfere with the activity of betel leaf extract.

[0011] In still another embodiment, the additive is selected fromnutrients such as proteins, carbohydrates and sugar, talc, magnesiumsterate, cellulose, calcium carbonate, starch-gelatin paste and/orpharmaceutically acceptable carriers.

[0012] In still another embodiment, the betel leaf extract isadministered orally or intramuscularly.

[0013] In yet another embodiment, the oral route is in the form ofcapsule, syrup, concentrate, powder or granules.

[0014] In yet another embodiment, the ratio of betel leaf extract to theadditive is in the range between 10 to 1.

[0015] In yet another embodiment, the betel leaf extract is administeredat a dosage level between 10 to 20 mg/kg of body weight for alternatedays for one month.

[0016] In yet another embodiment, the betel leaf extracts reduce theviability of L. donovani promastigotes in vitro by 57 to 79% and reducesplenic and liver parasite load by 93 to 95%.

[0017] In yet another embodiment of the present invention, the betelleaf extract or composition is used for the treatment of visceralleishmaniasis or kala-azar.

[0018] In yet another embodiment of the present invention, the betelleaf extract or lyophilized extract or composition is administeredtogether with or associated with a pharmaceutically acceptable additive.

[0019] In yet another embodiment of the present invention, the additiveis selected in such a manner it does not interfere with the activity ofbetel leaf extract.

[0020] In yet another embodiment of the present invention, the additiveis selected from nutrients such as proteins, carbohydrates, sugar andpharmaceutically acceptable carriers.

[0021] In yet another embodiment of the present invention, the betelleaf extract or the composition is administered orally orintramuscularly.

[0022] In still another embodiment, the oral route is in the form ofcapsule, syrup, concentrate, powder or granules.

[0023] In yet another embodiment of the present invention, the ratio ofbetel leaf extract to the additive is in the range between 10 to 1.

[0024] In still another embodiment of the present invention, the betelleaf extract is administered at a dosage level between 10 to 20 mg/kg ofbody weight for alternate days for one month.

[0025] In yet another embodiment of the present invention, the betelleaf extracts reduce the viability of L. donovani promastigotes in vitroby 57 to 79% and reduce splenic and liver parasite load by 93 to 95%.

[0026] In yet another embodiment of the present invention, a method oftreating visceral leishmaniasis or kala-azar by administering apharmaceutically acceptable composition of betel leaves extract orlyophilized extract.

[0027] In still another embodiment, the betel leaf extract is obtainedby crushing the betel leaf or extracting the crushed leafs with water ororganic solvents such as alcohol, carbontetrachloride, chloroform andacetone.

[0028] One more embodiment of the present invention relates to thepreparation of betel leaf extracts comprising the following steps;

[0029] washing of the fresh leaves of Piper betle and homogenizing in amixture blender;

[0030] sonicating in an ultrasonic bath with 2 to 3 bursts each for 15minutes and filtering the extract, if desired repeating the extractionat least once and drying; and

[0031] lyophilizing the extract to get a semi-solid mass

[0032] In another embodiment of the invention, the betel leaf (Piperbetle) is selected from Wild type, Climber type, Bangla type and Sweettype.

[0033] Effect of Betel Leaf Extract on the Viability of L. donovaniPromastigotes in vitro

[0034] Extract prepared from wild type, Bangla type, and sweet typebetel leaves are almost equally effective in reducing the viability ofL. donovani promastigotes in a dose dependent manner (Table 1). Extractsfrom wild type betel leaf at a final concentration of 12-mg/ml reducethe viability of L. donovani promastigotes by 79%. On the other hand,extract from Bangla type or sweet type betel leaf at the sameconcentration i.e. 12 mg/ml reduces promastigote viability by 57.5% and68.9% respectively. Thus, wild type betel leaf extract appears to haveslightly more anti leishmanial activity than other type of betel leafextract.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0035]FIG. 1: Photomicrographs of Glemsa stained liver and spleen smearsof L.donovani infected golden hamsters after treatment with PBS andbetel leaf extract.

[0036] The following examples are given by way of explanation and forillustration only and these examples should not be construed in anymanner to limit the scope of the invention.

EXAMPLES Example 1

[0037] 34.14 gm of fresh leaves of Piper betle thoroughly washed insterile water was homogenized with 100 ml of glass distilled water in amixture-blender. It was then sonicated in an ultrasonic bath with 3burst each for 15 min. The extract was filtered through Whatman No.1filter paper and the filtrate was collected. This process of extractionwas repeated three times. The combined extract was lyophilized yieldinga semi-solid mass weighing 1.17 gm. This was then tested for biologicalactivity.

Example 2

[0038] The fresh leaves of Piper betle weighing 21.68 gm homogenizedwith distilled water (60 ml) in a mixture—blender and then sonicated inan ultrasonic bath with 2 burst each for 15 min. It was allowed to beextracted overnight or 16 hours. Filtering through Whatman No.1 filterpaper separated the material extracted in water. This type of treatmentfor extraction was repeated for three times. The combined extract wasevaporated to dryness in a flash evaporator under reduced pressure at45° C. The residual substance was then dried in a desiccator under highvacuum and the semi-solid mass weighing 0.59 gm was tested forbiological activity.

[0039] Properties of the Materials:

[0040] The biologically active material obtained by examples 1 and 2 hasthe following properties:

[0041] i. The dried semisolid prepared as stated above was a darkcolored material soluble in water and dimethyl sulfoxide.

[0042] ii. Thin layer chromatography of the active material shows fivespots having R_(f) 0.75, 0.64, 0.50, 0.40 and 0.33 in the solvent systemof n-butanol, acetic acid and water in the ratio of 9:5:7 respectively.

[0043] iii. The HPLC analysis of the active material using IntersilODS-3 (4.6×250 mm) analytical column, solvent system methanol and waterin the ratio of 4:1 and a flow rate of 1.0 ml/min., detection at 217 nmresolved the material into eleven peaks with the retention time of 2.69,4.27, 5.95, 6.97, 7.49, 9.39, 11.20, 12.40, 15.53, 18.90 and 21.49 min.

Example 3

[0044] Parasite: L. donovani strain AG83 was originally obtained from anIndian Kala-azar Patient (Ghosh, A. K., Bhattacharaya, F. K. & Ghosh, D.K. 1985. Leishmania donovani: amastigote inhibition and mode of actionof berberine. Experimental Parasitology, 60: 404-13) and maintained ingolden hamsters. Amastigotes were isolated from spleens of L. donovaniinfected golden hamsters as described (Jaffe, C. L., Grimaldi, G. &Mcmohan-Pratt, D. 1984. In genes and Antigens of parasite: A Laboratorymanual, 2^(nd) ed_(n) [moral, C. M., Ed.], P.47, Rio de Janiero). Thespleen was rinsed in ice cold PBS-glucose (55 mm)/EDTA (2 mm), thenlightly homogenized, macroscopic particles were allowed to settle, andthe turbid suspension was decanted. This suspension was centrifuged at100 g for 10 min at 4° C. The amastigote-enriched suspension wascentrifuged at 800 g for 10 min. The pellet was suspended in 45% percoll (8.0 ml), and finally 25% per coll (4.0 ml) was layered over theamastigote suspension and centrifuged at 5000 g for 1 hr. The bandcontaining amastigote was then taken and washed with PBS, and finallyre-suspended in medium-199 (Gibco Laboratories, New York, N.Y., U.S.A.)supplemented with 20% FBS. Promastigotes were obtained by transformingamastigotes and were maintained in vitro in Medium-199 supplemented with8% FBS.

Example 4

[0045] In vitro growth of L. donovani promastigote in the presence ofbetel leaf extract 0.5×10⁶ L. donovani promastigotes in a total volumeof 250 μl M-199+10% FBS were incubated with graded concentrations ofbetel leaf extract for 24 hr at 22° C. Cells were then checkedmicroscopically for viability.

[0046] Effect of Betel Leaf Extract on the Viability of L. donovaniPromastigotes in vitro

[0047] Water extract is prepared from the following types of betel leaf(Piper betle)

[0048] 1. Wild type

[0049] 2. Climber type

[0050] 3. Bangla type

[0051] 4. Sweet type

[0052] Extract prepared from wild type, Bangla type, and sweet typebetel leaves are almost equally effective in reducing the viability ofL. donovani promastigotes in a dose dependent manner (Table 1). Extractsfrom wild type betel leaf at a final concentration of 12-mg/ml reducethe viability of L. donovani promastigotes by 79%. On the other hand,extract from Bangla type or sweet type betel leaf at the sameconcentration i.e. 12 mg/ml reduces promastigote viability by 57.5% and68.9% respectively. Thus wild type betel leaf extract appears to haveslightly more antileishmanial activity than other type of betel leafextract. TABLE 1 Betel leaf extracts reduce the viability of L. donovanipromastigotes in vitro Incubated with Percent reduction Medium Dose(mg/ml) of viability Wild type betel leaf extract — — 12.00 79.0 8.067.0 4.0 57.0 Bangla type betel leaf 12.0 65.3 extract 8.0 60.0 4.0 57.5Sweet type betel leaf 12.0 68.9 extract 8.0 63.8 4.0 63.8

Example 5

[0053] Antileishmanial Activity of Betel Leaf Extract in vivo

[0054] In vivo determination of antileishmanial activity of betel leafextract in golden hansters. Golden hamsters (4-6 weeks old) wereinfected by intracardiac injection of freshly prepared L. donovaniamastigotes (1×10⁷/hamster). One day post-infected hamsters were dividedinto three groups (5 animals per group); one group receivedintramuscular injection of wild type betel leaf extract (10 mg/kg bodyweight every other day), each animal received a total of 15 injections.Animals of the group received wild type betel leaf extract through oralroute at the same dose (10-mg/kg body weight every other day. Animals inthe control group received PBS by oral feeding every other day. Animalsof all groups were killed one week after the last treatment i.e. 5 weekspost infection period. The splenic and liver parasite load wasdetermined from impression smears after Giemsa staining. Results areexpressed as the total parasite load per organ using the formula:

Organ weight in mg×the number of amastigotes per cell nucleus×[2×10⁵]

[0055] Betel leaf extract at a concentration of 10 mg/kg body weight waseffective in reducing splenic and liver parasite load of L. donovaniinfected hamsters. Both routes i.e. intramascular and oral were equallyeffective. The type percent reduction of parasite burden varied between93 to 95% using these two routes. The results were shown in table-2 andFIG. 1. TABLE 2 Treatment of L. donovani infected golden hamsters withbetel leaf extracts. Route of Total parasite load (×10⁷) Treatment withadministration LIVER SPLEEN PBS Oral 82.6 ± 63.1* 10.6 ± 6.9  Wild typebetal leaf Oral 4.5 ± 3.0  0.7 ± 0.3 extract Intramuscular 3.7** 0.6 ±0.5

[0056] As shown in Table-2 and FIG. 1, betel leaf extract at aconcentration of 10 mg/kg body weight was effective in reducing splenicand liver parasite load of L. donovani infected hamsters. Of note, bothroutes i.e. intramascular or oral were almost equally effective. Thetype percent reduction of parasite burden varied between 93 to 95% usingthese two routes.

1. A method of treating visceral leishmaniasis or kala-azar in animalsincluding human beings said method comprising administering apharmaceutically effective amount of betel leaf extract or a compositioncomprising betel leaf extract to the animal.
 2. A method as claimed inclaim 1 wherein, the composition comprising effective amount of betelleaf extract together with or associated with a pharmaceuticallyacceptable additive.
 3. A method as claimed in claim 1 wherein, theadditive is selected from nutrients such as proteins, carbohydrates,sugar, talc, magnesium sterate, cellulose, calcium carbonate,starch-gelatin paste and/or pharmaceutically acceptable carriers,excipient, diluent or solvent.
 4. A method as claimed in claim 1wherein, the betel leaf extract or the composition comprising betelleafs extract is administered orally or intramuscularly.
 5. A method asclaimed in claim 1 wherein, the oral route is in the form of capsule,syrup, concentrate, powder or granules.
 6. A method as claimed in claim1 wherein, the ratio of betel leaf extract to the additive is in therange between 1-10:10-1.
 7. A method as claimed in claim 1 wherein, thebetel leaf extract is obtained by crushing the betel leaf or extractingthe crushed leafs with water or organic solvents such as alcohol,carbontetrachloride, chloroform and acetone.
 8. A method as claimed inclaim 1 wherein, the betel leaf extract or the composition comprisingbetel leafs extract is administered at a dosage level between 10 to 20mg/kg of body weight for alternate days for one month.
 9. A method asclaimed in claim 1 wherein, the betel leaf extract or the compositioncomprising betel leaf extract reduces the viability of L. donovanipromastigotes in vitro by 57 to 79% and reduce splenic and liverparasite load by 93 to 95%.
 10. A pharmaceutical composition useful forthe treatment of visceral leishmaniasis or kala-azar in animalsincluding human beings, said composition comprising effective amount ofbetel leaf extract together with or associated with a pharmaceuticallyacceptable additive.
 11. A composition as claimed in claim 10 wherein,the additive is selected in such a manner it does not interfere with theactivity of betel leaf extract.
 12. A composition as claimed in claim 10wherein the additive is selected from nutrients such as proteins,carbohydrates, sugar, talc, magnesium sterate, cellulose, calciumcarbonate, starch-gelatin paste and/or pharmaceutically acceptablecarriers.
 13. A composition as claimed in claim 10 wherein, thecomposition is administered orally or intramuscularly.
 14. A compositionas claimed in claim 10 wherein, the oral route is in the form ofcapsule, syrup, concentrate, powder or granules.
 15. A composition asclaimed in claim 10 wherein, the ratio of betel leaf extract to theadditive is in the range between 1-10:10-1.
 16. A composition as claimedin claim 10 wherein, the composition is administered at a dosage levelbetween 10 to 20 mg/kg of body weight for alternate days for one month.17. A composition as claimed in claim 10 wherein, the composition reducethe viability of L. donovani promastigotes in vitro by 57 to 79% andreduce splenic and liver parasite load by 93 to 95%.
 18. A compositionas claimed in claim 10 wherein, the betel leaf extract used is havingthe following properties i) The dried sample is a dark colored materialsoluble in water and dimethyl sulfoxide, ii) Thin layer chromatographyof the active material shows five spots having R_(f) 0.75, 0.64, 0.50,0.40 and 0.33 in the solvent system of n-butanol, acetic acid and waterin the ratio of 9:5:7 respectively, and iii) The HPLC analysis of theactive material using Intersil ODS-3 (4.6×250 mm) analytical column,solvent system methanol and water in the ratio of 4:1 and a flow rate of1.0 ml/min., detection at 217 nm resolved the material into eleven peakswith the retention time of 2.69, 4.27, 5.95, 6.97, 7.49, 9.39, 11.20,12.40, 15.53, 18.90 and 21.49 min.
 19. Use of betel leaf extract or acomposition comprising betel leaf extract for the treatment of visceralleishmaniasis or kala-azar in animals including human beings.
 20. Use asclaimed in claim 19 wherein, the betel leaf extract is administeredtogether with or associated with a pharmaceutically acceptable additive.21. Use as claimed in claim 19 wherein, the additive is selected in sucha manner that does not interfere with the activity of betel leafextract.
 22. Use as claimed in claim 19 wherein, the additive isselected from nutrients such as proteins, carbohydrates, sugar, talc,magnesium sterate, cellulose, calcium carbonate, starch-gelatin pasteand/or pharmaceutically acceptable carriers.
 23. Use as claimed in claim19 wherein the betel leaf extract or the composition is administeredorally or intramuscularly.
 24. Use as claimed in claim 19 wherein, theratio of betel leaf extract to the additive is in the range between1-10:10-1.
 25. Use as claimed in claim 19 wherein, the betel leafextract or composition is administered at a dosage level between 10 to20 mg/kg of body weight for alternate days for one month.
 26. Use asclaimed in claim 19 wherein, the betel leaf extract or compositionreduce the viability of L. donovani promastigotes in vitro by 57 to 79%and reduce splenic and liver parasite load by 93 to 95%.